The objective of this research is to develop novel influenza vaccines that overcome the limitations associated with current vaccines, i.e., suboptimal immunogenicity or suboptimal protection. Influenza viruses are a major, worldwide human health threat that demands vaccines with optimal immunogenicity and attenuation. No licensed human vaccines are yet available for highly pathogenic avian H5N1 viruses and studies conducted to date with inactivated H5N1 vaccines suggest that these test vaccines are of low immunogenicity. Our goals, therefore, are to develop live influenza vaccines that are more attenuated than the current cold-adapted vaccines but still induce protective immunity, to establish nanoemulsions as novel adjuvants for influenza vaccines, and to manufacture a master seed virus for use in clinical trials. In Aim 1, we plan to establish neuraminidase-deficient viruses as live attenuated influenza vaccines. A live attenuated influenza vaccine does exist but is restricted to a limited age group;alternative live attenuated influenza vaccines are, therefore, needed. Our preliminary studies show that viruses that lack the neuraminidase (NA) protein are attenuated, but provide protective immunity against challenge with lethal doses of influenza viruses. To further evaluate this approach, we propose to test the attenuation levels and protective efficacies of NA-deficient viruses of the H3N2 and H5N1 subtypes, representing 'seasonal influenza'and potentially pandemic influenza viruses. Adjuvants are often needed to improve the immunogenicity of influenza vaccines for unprimed individuals. Yet only a limited number of adjuvants are licensed for use in humans. In Aim 2, we therefore propose to establish nanoemulsions as adjuvants for influenza virus vaccines. Nanoemulsions are water-in-vegetable oil formulations with both virucidal and adjuvant properties. Our preliminary data with these formulations support their potential as mucosal adjuvants for influenza vaccines. We, therefore, plan to test these nanoemulsions as adjuvants for influenza viruses. The development of novel vaccines requires the transition from basic research to manufacturing of seed lots for (pre-)clinical testing, which often presents obstacles. This application takes advantage of the close collaboration of basic research groups and a state-of-the art manufacturing facility for biotherapeutics for pre-clinical animal studies and Phase I and II human clinical trials. In Aim 3, we propose to manufacture an NA-deficient H3 virus under current Good Manufacturing Practices for further use in clinical trials. Clinical evaluation of this vaccine candidate (which is beyond the scope of this application) will establish the potential of this approach for the development of novel live attenuated influenza virus vaccines and lay the foundation for the development of NA-deficient H5 virus vaccines for pandemic situations.